Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, a high morbidity disease worldwide with an estimated 106 million cases annually. N. gonorrhoeae infects the male and female genital tract and can often evade host immune mechanisms to persist until antibiotic intervention. The alarming increase in antibiotic resistant strains of N. gonorrhoeae, the often-asymptomatic nature of this disease in women, and the lack of a vaccine directed at crucial virulence determinants, speaks to the urgent need to study this pathogen during natural disease in humans to guide new therapies to treat infection. The studies proposed here are designed to identify gonococcal in vivo expressed factors (IVEFs) with the long- term goal to develop these as new therapeutic targets. Our studies have focused on a cohort of subjects attending the National Center for STD Control (NCSTD) in Nanjing, China due to the high level of disease in this region and reports of gonococcal strains with increased resistance. Using urethral and vaginal lavage samples obtained from the Nanjing cohort, together with RNA deep sequencing we were the first to identify genes expressed during natural infection, which were not detected in the corresponding gonococcal strains cultured under in vitro conditions. Within the group of common gonococcal genes detected with high-level expression during natural mucosal infection in men and women, was a group of previously uncharacterized gonococcal phage and hypothetical genes as well as genes encoding antimicrobial resistant determinants. We hypothesize that the gonococcus expresses genes in vivo that have not previously been identified in vitro. Despite differences in the gonococcal transcriptome expressed during natural infection in female and male subjects, common genes can be identified and have the potential to represent new therapeutic targets. We propose the following Aims to address this hypothesis: Aim 1. To define the N. gonorrhoeae transcriptome expressed during in vivo infection of the male and female genital tract and identify gonococcal IVEFs. Aim 2. To define the role of subsets of IVEF in N. gonorrhoeae pathogenesis. The studies proposed in this application are the first to examine the global transcriptome of N. gonorrhoeae during natural mucosal infection and to identify and characterize novel genes expressed in vivo that have not been yet hypothesized to exist.